Astrocyte Activation in the ACC Contributes to Comorbid Anxiety in Chronic Inflammatory Pain and Involves in The Excitation-Inhibition Imbalance.

Neurons within the anterior cingulate cortex (ACC) orchestrate the co-occurrence of chronic pain and anxiety. The ACC hyperactivity plays a crucial role in the emotional impact of neuropathic pain. Astrocyte-mediated neuroinflammatory is responsible for regulating the balance between excitation-inhibition (E/I) in the brain. However, there is limited understanding of the possible contributions of astrocytes in the ACC to comorbidity of anxiety and chronic inflammatory pain. This paper aims to investigate the possible contribution of astrocytes in the ACC to the comorbidity between anxiety and chronic inflammatory pain, as well as their involvement in the E/I imbalance of pyramidal cells. Our results show that CFA rats displayed allodynia and anxiety-like behaviors. The E/I balance in the ACC shifts to excitement in comorbidity of chronic pain and anxiety by western blotting, and electrophysiological recording. Result of RNA-Seq also indicated that E/I imbalance and neuroinflammation of ACC were involved in pain-anxiety comorbidity. Then, positive cells of GFAP but not Iba1 in the contralateral ACC were increased; the mRNA expression of GFAP and its activation-related proinflammatory cytokines (TNF-α, IL-6, and IL-1ß) in the contralateral ACC were also elevated. Furthermore, specific chemogenic inhibition of ACC astrocytes reversed comorbid pain and anxiety and suppressed high ACC excitability. Our data suggest that astrocytes participate in comorbid pain and anxiety and excitation-inhibition imbalance in ACC. Inhibition astrocyte activation can reduce anxiety related to pain and restore the imbalance in the ACC. These findings shed light on the involvement of astrocytes in comorbid conditions, offering valuable insights into a potential therapeutic approach for the co-occurrence of chronic pain and anxiety.

A neural circuit associated with anxiety-like behaviors induced by chronic inflammatory pain and the anxiolytic effects of electroacupuncture.

AIMS: Negative emotions induced by chronic pain are a serious clinical problem. Electroacupuncture (EA) is a clinically proven safe and effective method to manage pain-related negative emotions. However, the circuit mechanisms underlying the effect of EA treatment on negative emotions remain unclear. METHODS: Plantar injection of complete Freund's adjuvant (CFA) was performed to establish a rat model of chronic inflammatory pain-induced anxiety-like behaviors. Adeno-associated virus (AAV) tracing was used to identify excitatory synaptic transmission from the rostral anterior cingulate cortex (rACC) to the dorsal raphe nucleus (DRN). Employing chemogenetic approaches, we examined the role of the rACC-DRN circuit in chronic pain-induced anxiety-like behaviors and investigated whether EA could reverse chronic pain-induced dysfunctions of the rACC-DRN circuit and anxiety-like behaviors. RESULTS: We found that chemogenetic activation of the rACC-DRN circuit alleviated CFA-induced anxiety-like behaviors, while chemogenetic inhibition of the rACC-DRN circuit resulted in short-term CFA-induced anxiety-like behaviors. Further research revealed that the development of CFA-induced anxiety-like behaviors was attributed to the dysfunction of rACC CaMKII neurons projecting to DRN serotonergic neurons (rACCCaMKII-DRN5-HT neurons) but not rACC CaMKII neurons projecting to DRN GABAergic neurons (rACCCaMKII-DRNGABA neurons). This is supported by the findings that chemogenetic activation of the rACCCaMKII-DRN5-HT circuit alleviates anxiety-like behaviors in rats with chronic pain, whereas neither chemogenetic inhibition nor chemogenetic activation of the rACCCaMKII-DRNGABA circuit altered CFA chronic pain-evoked anxiety-like behaviors in rats. More importantly, we found that EA could reverse chronic pain-induced changes in the activity of rACC CaMKII neurons and DRN 5-HTergic neurons and that chemogenetic inhibition of the rACCCaMKII-DRN5-HT circuit blocked the therapeutic effects of EA on chronic pain-induced anxiety-like behaviors. CONCLUSIONS: Our data suggest that the reversal of rACCCaMKII-DRN5-HT circuit dysfunction may be a mechanism underlying the therapeutic effect of EA on chronic pain-induced anxiety-like behaviors.

Specificity for the correlation between the body surface and viscera in the pathological state of COPD: A prospective, controlled, and assessor-blinded trial.

Background: The association between the body surface and viscera remains obscure, but a better understanding of the body surface-viscera correlation will maximize its diagnostic and therapeutic values in clinical practice. Therefore, this study aimed to investigate the specificity of body surface-viscera correlation in the pathological state. Methods: The study subjects included 40 participants with chronic obstructive pulmonary disease (COPD) in the COPD group and 40 age-matched healthy participants in the healthy control group. Laser Doppler flowmetry, infrared thermography, and functional near-infrared spectroscopy were respectively adopted to measure 1) the perfusion unit (PU), 2) temperature, and 3) regional oxygen saturation (rSO2) of four specific sites distributed in the heart and lung meridians. These three outcome measures reflected the microcirculatory, thermal, and metabolic characteristics, respectively. Results: Regarding the microcirculatory and thermal characteristics of the body surface, the PU and temperature of specific sites on the body surface [i.e., Taiyuan (LU9) and Chize (LU5) in the lung meridian] in the COPD group were significantly increased compared with healthy controls (p < 0.05), whereas PU and temperature of other sites in the heart meridian [i.e., Shenmen (HT7) and Shaohai (HT3)] did not change significantly (p > 0.05). Regarding the metabolic characteristics, rSO2 of specific sites in the lung meridian [i.e., Taiyuan (LU9) and Chize (LU5)] and Shaohai (HT3) of the heart meridian in the COPD group was significantly decreased compared with healthy controls (p < 0.01), whereas rSO2 of Shenmen (HT7) in the heart meridian did not change significantly (p > 0.05). Conclusion: In the disease state of COPD, the microcirculatory, thermal, and metabolic characteristics of specific sites on the body surface in the lung meridian generally manifest more significant changes than those in the heart meridian, thereby supporting relative specificity for the body surface-viscera correlation in the pathological state.

Satellite glial cells drive the transition from acute to chronic pain in a rat model of hyperalgesic priming.

Chronic pain is one of the most common clinical syndromes affecting patients' quality of life. Regulating the transition from acute to chronic pain is a novel therapeutic strategy for chronic pain that presents a major clinical challenge. However, the mechanism underlying pain transitions remains poorly understood. A rat hyperalgesic priming (HP) model, which mimics pain transition, was established decades ago. Here, this HP model and RNA sequencing (RNA-seq) were used to study the potential role of neuroinflammation in pain transition. In this study, HP model rats developed prolonged hyperalgesia in the hind paw after carrageenan (Car) and PGE2 injection, accompanied by obvious satellite glial cell (SGC) activation in the dorsal root ganglion (DRG), as indicated by upregulation of GFAP. RNA-Seq identified a total of differentially expressed genes in the ipsilateral DRG in HP model rats. The expression of several representative genes was confirmed by real-time quantitative PCR (qPCR). Functional analysis of the differentially expressed genes indicated that genes related to the inflammatory and neuroinflammatory response showed the most significant changes in expression. We further found that the expression of the chemokine CXCL1 was significantly upregulated in the rat DRG. Pharmacological blockade of CXCL1 reduced protein kinase C epsilon overproduction as well as hyperalgesia in HP rats but did not prevent the upregulation of GFAP in the DRG. These results reveal that neuroinflammatory responses are involved in pain transition and may be the source of chronic pain. The chemokine CXCL1 in the DRG is a pivotal contributor to chronic pain and pain transition in HP model rats. Thus, our study provides a putative novel target for the development of effective therapeutics to prevent pain transition.

Dorsal root ganglia P2X4 and P2X7 receptors contribute to diabetes-induced hyperalgesia and the downregulation of electroacupuncture on P2X4 and P2X7.

Diabetic neuropathic pain (DNP) is highly common in diabetes patients. P2X receptors play critical roles in pain sensitization. We previously showed that elevated P2X3 expression in dorsal root ganglion (DRG) contributes to DNP. However, the role of other P2X receptors in DNP is unclear. Here, we established the DNP model using a single high-dose streptozotocin (STZ) injection and investigated the expression of P2X genes in the DRG. Our data revealed elevated P2X2, P2X4, and P2X7 mRNA levels in DRG of DNP rats. The protein levels of P2X4 and P2X7 in DNP rats increased, but the P2X2 did not change significantly. To study the role of P2X4 and P2X7 in diabetes-induced hyperalgesia, we treated the DNP rats with TNP-ATP (2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate), a nonspecific P2X1-7 antagonist, and found that TNP-ATP alleviated thermal hyperalgesia in DNP rats. 2 Hz electroacupuncture is analgesic against DNP and could downregulate P2X4 and P2X7 expression in DRG. Our findings indicate that P2X4 and P2X7 in L4-L6 DRGs contribute to diabetes-induced hyperalgesia, and that EA reduces thermal hyperalgesia and the expression of P2X4 and P2X7.

Analgesic effect of electroacupuncture on bone cancer pain in rat model: the role of peripheral P2X3 receptor.

Upregulation of P2X3 receptor (P2X3R) has been strongly implicated in nociceptive signaling including bone cancer pain (BCP). The present study, using rat bone cancer model, aimed to explore the role of P2X3R in regulating rat pain behavior under the intervention of electroacupuncture (EA). The BCP model was successfully established by injection with MRMT-1 breast cancer cell into the medullary cavity of left tibia for 3 × 104 cells/3 µL PBS in rats as revealed by obvious bone destruction, decreased paw withdrawal thresholds (PWTs), and reduced paw withdrawal latencies (PWLs). Western blot analyses showed that P2X3R expression was significantly upregulated in ipsilateral lumbar 4-6 (L4-6) dorsal root ganglia (DRG), but the difference not seen in spinal cord dorsal horn (SCDH). With the in-depth study of P2X3R activation, we observed that intrathecal injection of P2X3R agonist α,ß-meATP aggravated MRMT-1 induced BCP, while injection of P2X3R inhibitor A-317491 alleviated pain. Subsequently, we demonstrated that BCP induced mechanical allodynia and thermal hyperalgesia were attenuated after EA treatment. Under EA treatment, total P2X3R protein expression in ipsilateral DRGs was decreased, and it is worth mentioning that decreased expression of P2X3R membrane protein, which indicated that both the expression and membrane trafficking of P2X3R were inhibited by EA. The immunofluorescence assay showed that EA stimulation exerted functions by reducing the expression of P2X3R-positive cells in ipsilateral DRGs of BCP rats. Ca2+ imaging analysis revealed that the EA stimulation decreased the percentage of α,ß-meATP responsive neurons in DRGs and inhibited calcium influx. Notably, the inhibitory effect of EA on mechanical allodynia and nociceptive flinches was abolished by intrathecal injection of α,ß-meATP. These findings demonstrated EA stimulation ameliorated mechanical allodynia and thermal hyperalgesia in rat model of MRMT-1-induced BCP. EA exerts analgesic effect on BCP by reducing the overexpression and functional activity of P2X3R in ipsilateral DRGs of BCP rats. Our work first demonstrates the critical and overall role of P2X3R in EA's analgesia against peripheral sensitization of MRMT-1-induced BCP and further supports EA as a potential therapeutic option for cancer pain in clinic.

The Moxibustion-Induced Thermal Transport Effect Between the Heart and Lung Meridians With Infrared Thermography.

Objectives: By comparing the differences in the thermal transport effect between the heart and lung meridians induced by moxibustion, this study aimed to investigate the specificity of site-to-site associations on the body surface between different meridians. Methods: Eighty healthy participants were divided into the heart meridian intervention group and the lung meridian intervention group; moxibustion was performed at these two meridians, respectively. Baseline temperature and its change magnitude from baseline induced by moxibustion in 6 measuring sites of the heart and lung meridians were assessed by infrared thermography (IRT). Measuring sites included: Site 1 (Chize, LU5), Site 2 (midpoint of LU9 and LU5), Site 3 (Taiyuan, LU9), Site 4 (Shaohai, HT3), Site 5 (midpoint of HT7 and HT3), and Site 6 (Shenmen, HT7). Results: Forty participants (20 male and 20 female, 27.90 ± 0.52 years) were assigned to the heart meridian intervention group, and 40 participants (20 male and 20 female, 28.08 ± 0.54 years) were assigned to the lung meridian intervention group. In the lung meridian intervention group (moxibustion over LU5), the temperature of the distal sites in the lung meridian increased significantly at 5, 10, and 15 min compared with pre-moxibustion (P < 0.001). The temperature of Site 4 in the heart meridian, which was nearest to the moxibustion site, increased significantly compared with pre-moxibustion (P < 0.05), while the temperature in the distal sites of the heart meridian did not differ significantly during moxibustion. Regarding the comparison of temperature change magnitude from baseline (ΔT) between the two meridians, the ΔT of Site 2 in the lung meridian was significantly higher than Site 4 in the heart meridian at 5 and 10 min after moxibustion (P < 0.05), despite that Site 2 was more distal from the moxibustion site than Site 4. Similarly, the ΔT of Site 3 in the lung meridian was significantly higher than Site 5 and Site 6 in the heart meridian at 5, 10, and 15 min after moxibustion (P < 0.05). In the heart meridian invervention group, similar thermal transport effect between the two meridians was observed. The thermal transport effect of the distal sites along the heart meridian was more significant than that of the site closer to the moxibustion site but located in the lung meridian. Taken together, aforementioned results indicated that the moxibustion-induced thermal transport effect between the heart and lung meridians is generally more significant in the distal sites along the corresponding meridian than that in the closer sites of the other meridian. Conclusions: In the heart and lung meridians, the moxibustion-induced thermal transport effect is closely related to meridian routes, not just related to the absolute distance from the moxibustion site, thereby confirming the relative specificity of "site-to-site" associations on the body surface in these two meridians. Systematic Review Registration: https://clinicaltrials.gov/ct2/show/NCT05330403, identifier NCT05330403.

Electroacupuncture Ameliorates Mechanical Allodynia of a Rat Model of CRPS-I via Suppressing NLRP3 Inflammasome Activation in Spinal Cord Dorsal Horn Neurons.

Complex regional pain syndrome type-I (CRPS-I) is a chronic neurological disorder that results in severe pain and affects patients' life quality. Conventional therapies usually lack effectiveness. Electroacupuncture (EA) is an effective physical therapy for relieving CRPS-I pain. However, the mechanism underlying EA-induced analgesia on CRPS-I still remain unknown. Spinal NLRP3 inflammasome was recently identified to contribute to pain and neuroinflammation in a rat model of CRPS-I by our group. Here, we aimed to study whether EA could inhibit spinal NLRP3 inflammasome activation, thus resulting in pain relief and attenuation of spinal neuroinflammation in the rat model of CRPS-I. We established the rat chronic post-ischemic pain (CPIP) model to mimic CRPS-I. CPIP rats developed remarkable mechanical allodynia that could be relieved by daily EA intervention. NLRP3 inflammasome was activated in spinal cord dorsal horn (SCDH) of CPIP rats, accompanied with over-production of pro-inflammatory cytokine IL-1ß. Immunostaining revealed that the cellular distribution of NLRP3 was predominantly located in SCDH neurons. Pharmacological activation of NLRP3 inflammasome per se is sufficient to produce persistent mechanical allodynia in naïve animals, whereas blocking NLRP3 inflammasome attenuates mechanical allodynia of CPIP rats. EA exclusively reduced NLRP3 overexpression in SCDH neurons and attenuated spinal glial cell over-activation in CPIP rats. EA-induced anti-allodynia with attenuation of spinal glial cell over-activation were all mimicked by intrathecal blocking NLRP3 inflammasome and reversed by activating NLRP3 inflammasome, respectively, through pharmacological methods. Finally, spinal blocking IL-1ß attenuated mechanical allodynia and spinal glial cell over-activation in CPIP rats, resembling the effects of EA. In all, these results demonstrate that spinal NLRP3 inflammasome activation contributes to mechanical allodynia of the rat model of CRPS-I and EA ameliorates mechanical allodynia through inhibiting NLRP3 inflammasome activation in SCDH neurons. Our study further supports EA can be used as an effective treatment for CRPS-I.

Nrf2 Activation Mediates Antiallodynic Effect of Electroacupuncture on a Rat Model of Complex Regional Pain Syndrome Type-I through Reducing Local Oxidative Stress and Inflammation.

Complex regional pain syndrome type-I (CRPS-I) represents a type of neurovascular condition featured by severe pain in affected extremities. Few treatments have proven effective for CRPS-I. Electroacupuncture (EA) is an effective therapy for pain relief. We explored the mechanism through which EA ameliorates pain in a rat CRPS-I model. The chronic postischemic pain (CPIP) model was established using Sprague-Dawley rats to mimic CRPS-I. We found that oxidative stress-related biological process was among the predominant biological processes in affected hindpaw of CPIP rats. Oxidative stress occurred primarily in local hindpaw but not in the spinal cord or serum of model rats. Antioxidant N-acetyl cysteine (NAC) attenuated mechanical allodynia and spinal glia overactivation in CPIP model rats, whereas locally increasing oxidative stress is sufficient to induce chronic pain and spinal glia overactivation in naive rats. EA exerted remarkable antiallodynia on CPIP rats by reducing local oxidative stress via enhancing nuclear factor erythroid 2-related factor 2 (Nrf2) expression. Pharmacological blocking Nrf2 abolished antioxidative and antiallodynic effects of EA. EA reduced spinal glia overactivation, attenuated the upregulation of inflammatory cytokines, reduced the enhanced TRPA1 channel activity in dorsal root ganglion neurons innervating the hindpaws, and improved blood flow dysfunction in hindpaws of CPIP rats, all of which were mimicked by NAC treatment. Thus, we identified local oxidative injury as an important contributor to pathogenesis of animal CRPS-I model. EA targets local oxidative injury by enhancing endogenous Nrf2-mediated antioxidative mechanism to relieve pain and inflammation. Our study indicates EA can be an alternative option for CRPS-I management.

Exploring neuronal mechanisms involved in the scratching behavior of a mouse model of allergic contact dermatitis by transcriptomics.

BACKGROUND: Allergic contact dermatitis (ACD) is a common skin condition characterized by contact hypersensitivity to allergens, accompanied with skin inflammation and a mixed itch and pain sensation. The itch and pain dramatically affects patients' quality of life. However, still little is known about the mechanisms triggering pain and itch sensations in ACD. METHODS: We established a mouse model of ACD by sensitization and repetitive challenge with the hapten oxazolone. Skin pathological analysis, transcriptome RNA sequencing (RNA-seq), qPCR, Ca2+ imaging, immunostaining, and behavioral assay were used for identifying gene expression changes in dorsal root ganglion innervating the inflamed skin of ACD model mice and for further functional validations. RESULTS: The model mice developed typical ACD symptoms, including skin dryness, erythema, excoriation, edema, epidermal hyperplasia, inflammatory cell infiltration, and scratching behavior, accompanied with development of eczematous lesions. Transcriptome RNA-seq revealed a number of differentially expressed genes (DEGs), including 1436-DEG mRNAs and 374-DEG-long noncoding RNAs (lncRNAs). We identified a number of DEGs specifically related to sensory neuron signal transduction, pain, itch, and neuroinflammation. Comparison of our dataset with another published dataset of atopic dermatitis mouse model identified a core set of genes in peripheral sensory neurons that are exclusively affected by local skin inflammation. We further found that the expression of the pain and itch receptor MrgprD was functionally upregulated in dorsal root ganglia (DRG) neurons innervating the inflamed skin of ACD model mice. MrgprD activation induced by its agonist ß-alanine resulted in exaggerated scratching responses in ACD model mice compared with naïve mice. CONCLUSIONS: We identified the molecular changes and cellular pathways in peripheral sensory ganglia during ACD that might participate in neurogenic inflammation, pain, and itch. We further revealed that the pain and itch receptor MrgprD is functionally upregulated in DRG neurons, which might contribute to peripheral pain and itch sensitization during ACD. Thus, targeting MrgprD may be an effective method for alleviating itch and pain in ACD.

Amygdalar κ-opioid receptor-dependent upregulating glutamate transporter 1 mediates depressive-like behaviors of opioid abstinence.

Opiates produce a strong rewarding effect, but abstinence from opiate use emerges with severe negative emotions. Depression is one of the most frequent emotion disorders associated with opiate abstinence, which is thought to be a main cause for relapse. However, neurobiological bases of such an aversive emotion processing are poorly understood. Here, we find that morphine abstinence activates κ-opioid receptors (KORs) by increasing endogenous KOR ligand dynorphin expression in the amygdala, which in turn facilitates glutamate transporter 1 (GLT1) expression by activation of p38 mitogen-activated protein kinase (MAPK). Upregulation of GLT1 expression contributes to opiate-abstinence-elicited depressive-like behaviors through modulating amygdalar glutamatergic inputs to the nucleus accumbens (NAc). Intra-amygdala injection of GLT1 inhibitor DHK or knockdown of GLT1 expression in the amygdala significantly suppresses morphine-abstinence-induced depressive-like behaviors. Pharmacological and pharmacogenetic activation of amygdala-NAc projections prevents morphine-abstinence-induced behaviors. Overall, our study provides key molecular and circuit insights into the mechanisms of depression associated with opiate abstinence.

Anxiolytic effect of GABAergic neurons in the anterior cingulate cortex in a rat model of chronic inflammatory pain.

Chronic pain easily leads to concomitant mood disorders, and the excitability of anterior cingulate cortex (ACC) pyramidal neurons (PNs) is involved in chronic pain-related anxiety. However, the mechanism by which PNs regulate pain-related anxiety is still unknown. The GABAergic system plays an important role in modulating neuronal activity. In this paper, we aimed to study how the GABAergic system participates in regulating the excitability of ACC PNs, consequently affecting chronic inflammatory pain-related anxiety. A rat model of CFA-induced chronic inflammatory pain displayed anxiety-like behaviors, increased the excitability of ACC PNs, and reduced inhibitory presynaptic transmission; however, the number of GAD65/67 was not altered. Interestingly, intra-ACC injection of the GABAAR agonist muscimol relieved anxiety-like behaviors but had no effect on chronic inflammatory pain. Intra-ACC injection of the GABAAR antagonist picrotoxin induced anxiety-like behaviors but had no effect on pain in normal rats. Notably, chemogenetic activation of GABAergic neurons in the ACC alleviated chronic inflammatory pain and pain-induced anxiety-like behaviors, enhanced inhibitory presynaptic transmission, and reduced the excitability of ACC PNs. Chemogenetic inhibition of GABAergic neurons in the ACC led to pain-induced anxiety-like behaviors, reduced inhibitory presynaptic transmission, and enhanced the excitability of ACC PNs but had no effect on pain in normal rats. We demonstrate that the GABAergic system mediates a reduction in inhibitory presynaptic transmission in the ACC, which leads to enhanced excitability of pyramidal neurons in the ACC and is associated with chronic inflammatory pain-related anxiety.

Molecular mechanisms of opioid tolerance: From opioid receptors to inflammatory mediators (Review).

Opioids are considered the most effective analgesics for the treatment of both acute and chronic pain. However, prolonged opioid use can induce a certain level of tolerance to its analgesic effects, leading to a reduction in its effectiveness, addiction and abuse. A better understanding of the mechanisms underlying opioid tolerance may provide insights into this phenomenon and aid in the development of novel methods to combat the side effects of opioid tolerance. The present review focused on two major contributors to tolerance, opioid receptors and inflammatory mediators. The molecular mechanisms involved in the desensitization of the opioid receptors were briefly described, including their phosphorylation, internalisation and recycling. Subsequently, the effects of Toll like receptor 4/NOD-like receptor family pyrin domain containing 3-mediated proinflammatory responses in opioid tolerance were discussed, aiming in supporting the identification of novel therapeutic targets.

Electroacupuncture Regulates Pain Transition Through Inhibiting PKCε and TRPV1 Expression in Dorsal Root Ganglion.

Many cases of acute pain can be resolved with few side effects. However, some cases of acute pain may persist beyond the time required for tissue injury recovery and transit to chronic pain, which is hard to treat. The mechanisms underlying pain transition are not entirely understood, and treatment strategies are lacking. In this study, the hyperalgesic priming model was established on rats to study pain transition by injection of carrageenan (Car) and prostaglandin E2 (PGE2). The expression levels of protein kinase C epsilon (PKCε) and transient receptor potential vanilloid 1 (TRPV1) in the L4-L6 dorsal root ganglion (DRG) were investigated. Electroacupuncture (EA) is a form of acupuncture in which a small electric current is passed between a pair of acupuncture needles. EA was administrated, and its effect on hyperalgesia and PKCε and TRPV1 expression was investigated. The PKCε-TRPV1 signaling pathway in DRG was implicated in the pain transition. EA increased the pain threshold of model animals and regulated the high expression of PKCε and TRPV1. Moreover, EA also regulated hyperalgesia and high TRPV1 expression induced by selective PKCε activation. We also found that EA partly increased chronic pain threshold, even though it was only administered between the Car and PGE2 injections. These findings suggested that EA could prevent the transition from acute to chronic pain by inhibiting the PKCε and TRPV1 expression in the peripheral nervous system.

Role of GABAAR in the Transition From Acute to Chronic Pain and the Analgesic Effect of Electroacupuncture on Hyperalgesic Priming Model Rats.

Chronic pain is a costly health problem that impairs health-related quality of life when not effectively treated. Regulating the transition from acute to chronic pain is a new therapeutic strategy for chronic pain that presents a major clinical challenge. The underlying mechanisms of pain transition are not entirely understood, and strategies for preventing this transition are lacking. Here, a hyperalgesic priming model was used to study the potential mechanism by which γ-aminobutyric acid receptor type A (GABAAR) in the dorsal root ganglion (DRG) contributes to pain transition. Furthermore, electroacupuncture (EA), a modern method of acupuncture, was administered to regulate pain transition, and the mechanism underlying EA's regulatory effect was investigated. Hyperalgesic priming was induced by intraplanar injection of carrageenan (Car)/prostaglandin E2 (PGE2). The decrease in mechanical withdrawal threshold (MWT) induced by PGE2 returned to baseline 4 h after injection in NS + PGE2 group, and still persisted 24 h after injection in Car + PGE2 group. Lower expression of GABAAR in the lumbar DRG was observed in the model rats. Furthermore, activating or blocking GABAAR could reversed the long-lasting hyperalgesia induced by Car/PGE2 injection or produced a persistent hyperalgesia. In addition, GABAAR may be involved in Protein Kinase C epsilon (PKCε) activation in the DRG, a mark molecular of pain transition. EA considerably increased the mechanical pain thresholds of hyperalgesic priming model mammals in both the acute and chronic phases. Furthermore, EA upregulated the expression of GABAAR and inhibited the activation of PKCε in the DRG. In addition, peripheral administration of picrotoxin blocked the analgesic effect of EA on the model rats and abolished the regulatory effect of EA on PKCε activation. These findings suggested that GABAAR plays a key role in both the transition from acute to chronic pain and the analgesic effect of EA on hyperalgesic priming.

Electroacupuncture Ameliorates Chronic Inflammatory Pain-Related Anxiety by Activating PV Interneurons in the Anterior Cingulate Cortex.

Chronic inflammatory pain is a common clinical disease that tends to be associated with negative emotions such as anxiety and depression. The anterior cingulate cortex (ACC) is involved in pain and pain-related anxiety, and γ-aminobutyric acid (GABA)-ergic interneurons play an important role in chronic pain and anxiety. Electroacupuncture (EA) has good analgesic and antianxiety effect, but the underlying mechanisms have not yet been fully elucidated. In this study, we established a chronic inflammatory pain model and observed that this model induced anxiety-like behaviors and decreased the numbers of parvalbumin (PV) and somatostatin (SOM) positive cells. Activation of PV but not SOM interneurons by chemogenetic techniques alleviated anxiety-like behaviors and pain sensation. EA treatment improved pain sensation, anxiety-like behaviors and increased the number of PV- positive cells in the ACC, but did not affect on the number of SOM-positive cells in the ACC. Moreover, specific inhibition of PV interneurons by chemogenetic methods reversed the analgesic and antianxiety effects of EA. These results suggest that EA ameliorates chronic inflammatory pain and pain-related anxiety by upregulating PV but not SOM interneurons in the ACC.

Electroacupuncture inhibits the interaction between peripheral TRPV1 and P2X3 in rats with different pathological pain.

Chronic pain is regarded to be one of the common and refractory diseases to cure in the clinic. One hundred Hz electroacupuncture (EA) is commonly used for inflammatory pain and 2 Hz for neuropathic pain possibly by modulating the transient receptor potential vanilloid subtype 1 (TRPV1) or the purinergic P2X3 related pathways. To clarify the mechanism of EA under various conditions of pathological pain, rats received a subcutaneous administration of complete Freund's adjuvant (CFA) for inflammatory pain and spared nerve injury (SNI) for neuropathic pain. The EA was performed at the bilateral ST36 and BL60 1 d after CFA or SNI being successfully established for 3 consecutive days. The mechanical hyperalgesia test was measured at baseline, 1 d after model establishment, 1 d and 3 d after EA. The co-expression changes, co-immunoprecipitation of TRPV1 and P2X3, and spontaneous pain behaviors (SPB) test were performed 3 d after EA stimulation. One hundred Hz EA or 2Hz EA stimulation could effectively down-regulate the hyperalgesia of CFA or SNI rats. The increased co-expression ratio between TRPV1 and P2X3 at the dorsal root ganglion (DRG) in two types of pain could be reduced by 100Hz or 2Hz EA intervention. While 100Hz or 2Hz EA was not able to eliminate the direct physical interaction between TRPV1 and P2X3. Moreover, EA could significantly inhibit the SPB induced by the co-activation of peripheral TRPV1 and P2X3. All results indicated that EA could significantly reduce the hyperalgesia and the SPB, which was partly related to inhibiting the co-expression and indirect interaction between peripheral TRPV1 and P2X3.

Phosphoproteomic Profiling of Rat's Dorsal Root Ganglia Reveals mTOR as a Potential Target in Bone Cancer Pain and Electro-Acupuncture's Analgesia.

Bone cancer pain (BCP) is a clinical refractory mixed pain involving neuropathic and inflammatory pain, with the underlying mechanisms remaining largely unknown. Electro-acupuncture (EA) can partly alleviate BCP according to previous research. We aim to explore the proteins and major pathways involved in BCP and EA treatment through phosphoproteomic profiling. BCP rat model was built by tibial inoculation of MRMT-1 mammary gland carcinoma cells. Mechanical hyperalgesia determined by paw withdrawal thresholds (PWTs) and bone destruction manifested on the radiographs confirmed the success of modeling, which were attenuated by EA treatment. The differentially expressed phosphorylated proteins (DEPs) co-regulated by BCP modeling and EA treatment in rat dorsal root ganglions (DRGs) were analyzed through PEX100 Protein microarray. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEPs were significantly enriched in mammalian target of rapamycin (mTOR) signaling pathway. The phosphorylations of mTOR at Ser2448 and Thr2446 were increased in BCP and downregulated by EA. In addition, the phosphorylation of S6K and Akt, markers of the mTOR complex, were also increased in BCP and downregulated by EA. Inhibition of mTOR signaling alleviated the PWTs of BCP rats, while the mTOR agonist impaired the analgesic effect of EA. Thus, our study provided a landscape of protein phosphorylation changes in DRGs of EA-treated BCP rats and revealed that mTOR signaling can be potentially targeted to alleviate BCP by EA treatment.

The interaction between P2X3 and TRPV1 in the dorsal root ganglia of adult rats with different pathological pains.

Peripheral inflammatory and neuropathic pain are closely related to the activation of purinergic receptor P2X ligand-gated ion channel 3 (P2X3) and transient receptor potential vanilloid 1 (TRPV1), but the interaction between P2X3 and TRPV1 in different types of pathological pain has rarely been reported. In this study, complete Freund's adjuvant (CFA)-induced inflammatory pain and spared nerve injury (SNI)-induced neuropathic pain models were established in adult rats. The interactions between P2X3 and TRPV1 in the dorsal root ganglion were observed by pharmacological, co-immunoprecipitation, immunofluorescence and whole-cell patch-clamp recording assays. TRPV1 was shown to promote the induction of spontaneous pain caused by P2X3 in the SNI model, but the induction of spontaneous pain behaviour by TRPV1 was not completely dependent on P2X3 in vivo. In both the CFA and SNI models, the activation of peripheral P2X3 enhanced the effect of TRPV1 on spontaneous pain, while the inhibition of peripheral TRPV1 reduced the induction of spontaneous pain by P2X3 in the CFA model. TRPV1 and P2X3 had inhibitory effects on each other in the inflammatory pain model. During neuropathic pain, P2X3 facilitated the function of TRPV1, while TRPV1 had an inhibitory effect on P2X3. These results suggest that the mutual effects of P2X3 and TRPV1 differ in cases of inflammatory and neuropathic pain in rats.